Contactless electropermeabilization electrode and method

ABSTRACT

Devices and methods for delivering an electropermeabilizing pulse of electric energy to a tissue surface to enable delivery into cells in the tissue therapeutic substances. The device incorporates a source capable of generating a sufficient voltage potential to deliver a spark across a gap and delivers same to the tissue surface.

CROSS-REFERENCE TO RELATED APPLICATIONS SECTION

This application is a divisional application of U.S. patent applicationSer. No. 13/263,802, filed Oct. 10, 2011, which is a 371 National stageentry of International Application No. PCT/US2010/031431, filed Apr. 16,2010, and claims the benefit of U.S. Provisional Application No.61/212,803, filed Apr. 16, 2009, the entire contents of each of whichare incorporated herein by reference.

FIELD OF THE DISCLOSURE

This disclosure relates to delivery of therapeutic substances includingmacromolecules, such as polynucleotides and polypeptides, into mammaliancells, particularly cells lying adjacent or otherwise near tissuesurfaces, using a novel electropermeabilization system.

BACKGROUND

The following description includes information that may be useful inunderstanding the present disclosure. It is not an admission that anysuch information is prior art, or relevant, to the presently claimedinventions, or that any publication specifically or implicitlyreferenced is prior art.

Electropermeabilization of mammalian cells is a technique that has beenused for delivery of therapeutic substances including small molecules,such as anticancer agents bleomycin and cisplatin, and macromoleculessuch as nucleic acids and proteins. Typically, delivery of suchsubstances into cells is brought about by injecting the substance intothe tissues containing the cells, which injection merely places suchsubstance into the interstitial spaces between the cells, followed byphysically contacting the tissues with a metallic electrode of oneconfiguration or another and applying an electric potential across theelectrodes. Usually, the electrode is manifest in the form of at leasttwo opposing needle-like tissue piercing rods or tubes comprising ananode and a cathode. Other forms of tissue contacting electrodes includenon-penetrating electrodes such as planar pads as found in “caliper”electrode devices such as disclosed in U.S. Pat. No. 5,439,440 andmeander electrodes such as disclosed in U.S. Pat. No. 6,009,345. Stillother electrode types have included minimally invasive electrodes suchas disclosed in U.S. Pat. No. 6,603,998.

With regard to the electrodes as mentioned above, all operate within aparadigm well understood in the electrical arts to require express anddirect contact between the electrode and the tissue undergoingelectropermeabilization. Further, the electrical potential placed acrossthe positive and negative poles, often expressed as “field strength” inVolts/centimeter, has been in the vicinity of tens to hundreds of voltsper centimeter, i.e., voltage potential between the positive andnegative poles spaced apart in, or on, the tissue a given distance.Typically, distances between electrodes are from tenths of centimetersto full centimeters of length. In most disclosures concerningelectropermeabilization, the voltages required to provide a fieldstrength sufficient for cell poration in the tissues are anywhere fromone Volt for skin tissues to upwards of five or six hundred volts forcells lying in deeper body tissues. The various levels of voltageapplied are typically dependent upon the spacing of the positive andnegative electrodes and the electric resistance of the tissue undergoingtreatment.

There have been many recent advances in the art ofelectropermeabilization wherein low voltage potentials have been appliedto skin tissues. In many of these cases, the low voltage applied hasbeen tied to very lengthy time periods for applying the electricalenergy. In some cases, the electrical energy has been applied in adirect current form understood in the arts as providing anelectrophoresis or iontophoresis effect wherein substances are movedthrough the tissue slowly. In such conditions and particularly withsmall molecules, the electric pulses only provide for the molecules tobe moved through tissue interstitial space, not inside the cells withinthe tissue. Even where the low voltage has been applied for shortperiods of time, the electrodes comprise the typical complex two polearray arrangement, i.e., for example, at least one each of independentlychargeable cathode(s) and anode(s) placed in contact with the tissues.Other recent disclosures discuss the use of very high voltages, in the10,000 plus Volt range, for very short time periods to achieve deliveryof substances into cells but none the less require contact of theelectrodes with the tissue.

Whether using low or high voltages, tissue contacting electrodes systemsare subject to practical limitations primarily concerning safety andcomfort, or lack thereof, to the mammal undergoing treatment. There isalso the practicality or impracticality of manufacturing complexminiaturized arrays containing both anodes and cathodes often organizedto be pulsed independently of one another in various sequences anddirection of pulsing. Use of high voltages with tissue piercing andsurface touching electrodes can be dangerous for the potential of severeelectric shock if conditions include high amperage over a time greaterthan 10 millisec. Use of low voltages over an extended period of time,though typically not dangerous, has the potential of being uncomfortableto the patient mammal or otherwise requires complex manufacturingprocesses. In addition, there are concerns that voltage facilitatedsystems or delivery methods result in low levels of efficacy.

Still other issues are of concern in delivery of substances to skin ortissue surface cells. For example, some systems disclose methods ofdelivering the substance through the skin surface, i.e., the stratumcorneum, followed by delivery of the electric potential with the typicaltissue penetrating or surface contacting electrodes. With regard to suchsubstance delivery, instead of direct injection some systems attempt todraw the substance directly through the stratum corneum by iontophoresisand/or electrophoresis by applying various means to first ablate thestratum corneum before providing the substance and electric potential.For example, one system uses a laser beam to poke holes in the stratumcorneum (U.S. Pat. No. 6,527,716). Another uses a high intensity tissueablating spark not unlike a cauterizing surgical instrument (U.S. Pat.No. 6,611,706). In each of such systems, the methodology relies onphysically disrupting the stratum corneum in order to deliver thesubstance and further aid in the transmission of the electrical energyfrom the tissue contacting electrodes into the tissue.

Thus, there is in the art a need to advance delivery of substances intocells using electropermeabilization in a manner that avoids electricalhazards, discomfort to the treated patient, damage to the tissues, andcomplex manufacturing.

SUMMARY

Disclosed herein is a novel methodology and electropermeabilization (orelectroporation) device that delivers an electric potential having asufficient field strength for causing cell permeability (or reversiblepore formation) in skin-based or tissue surface-based cells withouteither causing potential hazardous electrical conditions, physicaldamage to the tissue surface, or noticeable discomfort. Further, thenovel electrode is capable of delivering the necessary electricpotential without being invasive, i.e., not penetrating the skin.

In one embodiment, there are disclosed methods of delivering therapeuticsubstances, which can include drugs, small molecules, and macromoleculesto cells associated with tissue surfaces of a mammal, such as that ofskin. With respect to skin, the current method can be used to deliversuch substances into cells of epidermal and dermal tissue. As definedherein, macromolecules include large steroidal chemical compounds, aswell as polynucleotides such as DNA, RNA, siRNA and the like, andpolypeptides such as proteins comprising chains of amino acids between 8and 3000 amino acid units. In an embodiment, polynucleotides includesingle and double stranded moieties, as well as both linear and circularpolynucleotide sequences encoding polypeptides comprising wholefunctional proteins and fragments thereof, including short epitopes.

In one embodiment, there are disclosed methods of imparting a sufficientelectric energy pulse (or electropermeabilizing electric voltagepotential) to a tissue surface to cause reversible poration in cells ofthe tissue for the cellular delivery of a therapeutic substance into theporated cells. In another embodiment, there are disclosed methods ofdelivering the pulse of electric energy (or delivering or dischargingthe electric voltage potential) without the need for a tissuepenetration, and preferably without contacting the tissue with theelectrode or “non-contact.” Thus, the electrode advances the art ofimparting electroporative electric energy pulses to tissues for thepurpose of causing electropermeabilization in that there need not be anytrauma to the tissue, such as penetrating the tissue by a needle-likemember, or alternatively, compressing, scratching, burning, or ablationof the skin surface other than the subcutaneous placementpreelectropermabilization of the substance to be delivered to the cells.

In still another embodiment, the electropermeabilization system advancesthe delivery of electric energy by providing a novel route toelectropermeabilization of cells for the direct delivery into the cellsof the therapeutic substances in that present implementations provide asimplification of the electrode circuitry from the prior art requirementof two electrodes of opposing polarities at the delivery site in atissue to instead, use of only a single electrode of a singular polarityat the delivery site that works in concert with (or otherwisedischarging against) the polarity of the quiescent or grounded tissue ofthe subject being treated whether that is more positive or more negativerelative to the delivering electrode polarity. In a further embodiment,the energy imparted to the tissue is dependent upon a combination of thepotential of the voltage discharged and the distance or “gap” betweenthe electrode tip and the tissue surface. In a related embodiment, thegap, rather than exist exclusively between the non-contact electrode andthe tissue surface, can include a “spark-gap regulator” element locatedbetween the non-contact electrode and the device circuitry. Thespark-gap regulator allows for discharge of the voltage potential in apredeterminable fashion limiting the amount of current relative to time.Furthermore, in this embodiment, the distal end of the electrode can bein contact with the surface of the tissue, i.e., can contact the surfaceof skin. In a related embodiment, the singular polarity of thenon-contact electrode allows for reference not to “field strength” as iscommon in prior electroporation systems requiring electrodes of opposingpolarity, but to “total energy” imparted to the tissue. In a furtherembodiment, the pulse of electric energy provided by the non-contactelectrode can have a total electrical charge imparted into the tissue ofbetween 2.8×10E-8 and 2.5×10E-6 Coulombs to achieveelectropermeabilization that is equivalent to between about 0.025 mJ and270 mJ (millijoules) of energy, as further disclosed in Table I.

In another embodiment, an alternative single polarity electrode maycomprise a tissue contacting array of noninvasive or alternativelyminimally invasive electrodes comprising needle-like projections, ineach case, all of a singular polarity. In an embodiment, the array canbe constructed from a single electrically conductive material. As usedherein a “single polarity electrode” or a “singular polarity electrode”is one that is constructed so as to possess only one pole, namely eitheranode or cathode, as the case may be. Where such a singular polarityelectrode comprises an array of needle like projections, all suchprojection electrodes are pulsed in one pole from the electric energygeneration source to the tissue. As used herein, a noninvasive electrodeis an electrode comprising an array of needle-like projections that donot penetrate through a stratum corneum layer of skin tissue. As usedherein, a minimally invasive electrode is an electrode array comprisingneedle-like projections that penetrate through a stratum corneum layerto a depth of subdermal tissue, i.e., about 1-2 mm. In this alternateembodiment, the electrode does contact tissue surface but energyimparted from the singular electrode derives from an electrical pulsedriven across a spark-gap regulator as disclosed herein.

In some embodiments where the electrodes are minimally invasive orcontact the surface of the tissue, the energy source can be one that iscapable of generating a sufficient electric voltage potential over aperiod of time less than 1 millisecond, and preferably less than 100microseconds. In preferred embodiments, the energy source is apiezoelectric crystal. In such embodiments, only one gap is necessarybetween the energy source and the tissue—such gap is between theelectrode's distal end and the tissue. In some embodiments, theelectrode can have a spark-gap regulator.

In another embodiment, there provided methods of delivering theelectroporative pulse of energy in a regulated manner wherein the totalenergy discharge is controlled by a “spark-gap regulator”. In anembodiment, the spark-gap regulator comprises an electrically neutral ornonconductive housing, such as clear plastic, encasing two electricleads separated by a “gap” of a predetermined measurement and of apredetermined electrical resistance rating and optionally under eitherpositive or negative atmosphere pressure conditions, such as for examplea vacuum or atmospheric pressure. In related embodiments, the spark-gapregulator provides the ability for the delivery system to be equippedwith any of a variety of spark-gap regulators each manufactured topreset voltage thresholds for use in setting predetermined deliveryparameters including such as minimum and peak voltages, current range,and total and/or net charges/energies (Coulombs or Joules) imparted intothe tissue. It is contemplated that each of these parameters can affectthe immune outcome for a particular disease or disorder being treated inthe mammal.

In still another embodiment, the spark-gap regulator can be constructedto provide for any number of electrical energy levels (or electricvoltage potentials) to be dischargable across the gap and therebyregulate the discharge of the voltage potential applied to the tissuevia the electrode. In some instances the electrodes include: contactingsingle polarity electrode array, or alternatively, the non-contactelectrode.

In yet other embodiments, the energy source for providing a singlepolarity potential can comprise any of a 1 to 12 V battery, a chargedcapacitor, a charge coil, a piezoelectric crystal, or a Van de Graaffgenerator.

In further embodiments, there is provided methods of eliciting an immuneresponse in a mammalian tissue by single polarityelectropermeabilization.

Other features and advantages will be apparent from the followingdrawings, detailed description, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofillustrative embodiments, is better understood when read in conjunctionwith the appended drawings. For the purpose of illustrating theembodiments, there are shown in the drawings example constructions ofthe embodiments; however, the embodiments are not limited to thespecific methods and instrumentalities disclosed. In the drawings:

FIG. 1 is a schematic drawing depicting examples of elements of a devicein accordance with the present disclosure;

FIG. 2 is a close up representation of the non-contact singular polarityelectrode separated by a “gap” from the tissue surface;

FIGS. 3A, B, and C are schematic depictions of three shapes that may beused for the non-contact singular polarity electrode;

FIGS. 4A and B are schematic drawings depicting examples of elements ofalternate designs of the device of FIG. 1;

FIG. 5 is a three dimensional view of a spark-gap regulator;

FIGS. 6A, B, and C are graphs depicting the pulse characteristics ofelectrostatic discharges from various energy sources;

FIGS. 7A to F are drawings of various example electric circuitsgenerally laying out the charge generation elements of alternateembodiments;

FIGS. 8A and B are graphs showing pulse discharges using a Van de Graaffgenerator; and

FIG. 9 is a graph showing titers of antibodies to influenza protein (NP)following dermal injection and pulsed using the spark-gap method of thedisclosure.

DETAILED IMPLEMENTATIONS OF THE DISCLOSURE

Provided herein are novel electropermeabilization (or electroporation)systems and methods for imparting electropermeabilizing electric pulsesof energy to surfaces of tissues using a non-contact electrode, whereinthe electrode does not contact the tissue, or a contact electrodewherein the electrode does contact the tissue with minimal penetrationof skin, and preferable only surface contact.

FIG. 1 is a schematic drawing depicting examples of elements of a device10 resting on a tissue surface 15 wherein there has been placed a bolusof therapeutic substance 16. The device has housing 11 containing anelectric energy source 14, circuitry 13 and non-contact singularpolarity electrode 12. The housing 11 forms a cowling around andextending a predetermined measurement greater than a length of theelectrode 12.

FIG. 2 is a close up representation of the non-contact singular polarityelectrode 12 separated by “gap” 17 from the tissue surface 15.

FIGS. 3A, B, and C are schematic depictions of three shapes that areuseful for the non-contact singular polarity electrode. In FIG. 3A, thetip of the electrode is spherical. In FIG. 3B, the tip is pointed. InFIG. 3C, the tip is flat.

The term “sufficient voltage potential” or “voltage potentialsufficient” when referring to the energy source and the voltagepotential such source generates, the term refers to a minimum amount ofvoltage potential that is required to generate a spark that is able tojump across the gap in the electrode (i.e., the space gap of thespark-gap regulator), or the gap between the electrode and the surfaceof the tissue when spark-gap regulator, to deliver a desired charge tothe tissue.

The term “predetermined distance” or “desired distance” when referringto the gap in the electropermeabilization system, refers to either thespace gap of the spark-gap regulator or the gap between the distal endof the electrode and the tissue surface (when no spark-gap regulator ispresent). However, if both the space gap and the gap between electrodeand tissue are provided in said system, neither gap will be larger thanthat of the predetermined distance. In embodiments where both gaps arepresent, the gap between the distal end of the electrode and the surfaceof the tissue will be the same or shorter than the space gap of thespark-gap regulator.

In a first embodiment, the device 10 can comprise an electrode 12 thatneed not contact the tissue surface 15 in order to impart into thetissue a pulse of electric energy sufficient to causeelectropermeabilization of cells in the tissue. This ability to providesufficient energy into the tissue without the electrode 12 touching thetissue 15 is a novel concept throughout the field of the electroporativearts. Moreover, the energy can be supplied to the tissue via theelectrode 12 without causing any discernable damage, such as burning orablation of the tissue surface 15. Rather, as concerns imparting energyto the stratum corneum, for example, the energy is imparted to thetissue with the stratum corneum remaining macroscopically intact.Specifically, the electric energy is applied directly to the tissuesurface 15 at a location directly above or otherwise in the vicinity ofa previously delivered bolus of therapeutic substance that has beeninjected just under the tissue surface 15. In an embodiment, the bolusis injected into a mammalian patient, such as a human, at any ofsubepidermal, subcutaneous, or intradermal locations. The electricdischarge can be directed as single, or alternatively, a plurality ofindividual pulses to various portions of the tissue area containing thebolus. By macroscopically intact, it is meant that there is nodiscernable macroscopic histological damage or alteration to the stratumcorneum.

In this first embodiment, the non-contact electrode 12 operates as apoint discharge source wherein electric energy pulsed to the electrodefrom the system circuitry is discharged to the tissue surface 15 acrossa space (air) gap 17 of a predetermined measure. Specifically, theelectric pulse is discharged via a spark jumping across the gap 17 intothe tissue after which the electric energy dissipates through thetissue. Thus, the electrode 12 imparts a singular polarity staticdischarge into the tissue. The energy pulses can comprise from 1 to over100 individual sparks imparted to the tissue surface 15 with theelectrode tip resting from anywhere of 0.01 cm to 10 mm above the tissuesurface. In an embodiment, the total energy passed to the tissues viathe spark is sufficient to cause electropermeabilization of cells withinthe tissue but is not so great as to cause any discernable damage to thetissue or the tissue surface 15. Generally, the electric energy can bedescribed as static electric pulses. Typically, in order to achievetransfer of the energy pulse from the electrode across an air gap 17 tothe tissue surface 15 requires voltage potentials that are in the rangeof kilovolts. Although high voltage potentials alone suggest thepossibility of danger or discomfort potential for the treated mammal,since the generation of high voltages is of a static electric nature,and because the discharge across the air gap 17 occurs in an extremelyshort time frame (nanoseconds), there is little current generated in thetissue to cause tissue damage despite amperages reaching meaningfulvalues. Thus, the system provides for discharge of high voltagepotential with little danger of injury to the patient. In an embodiment,the voltage potential can range from about between 5 kVolts to over 100kVolts. The time frame of discharge can be about between 5 nanosecondsto 5 microseconds where the gap 17 between electrode and tissue surfaceis between 0.01 cm and 1 cm. Where, as in this embodiment, the gap 17between electrode 12 and tissue surface 15 acts as a defacto spark-gapregulator, i.e., there is no separate spark-gap regulator in theupstream circuitry, the gap 17 can be up to 1 cm without the resultantcurrent flow becoming too great for both safety and sensation concerns.Within this gap range, in each instance the imparting of electric energyis barely perceptible.

Various sources of electric energy can be used to generate the voltagepotentials for the non-contact electrode 12. For example, a Van deGraaff apparatus can be used to generate static electric potentialswherein a metal dome acts as a capacitor for building charge of between5 kVolts and 100 kVolts that can be channeled through the circuitry 13to the electrode for a discharge period of between 5 nanoseconds and 5microseconds. Alternatively, a piezoelectric crystal can be used whereinan impact mechanism can create high voltage short duration pulses of 20to 100 nanoseconds. In yet another alternative, a high voltage Teslacoil with a transformer and switch can be arranged wherein a primarycoil can be used to induce a secondary coil to a 5 kVolts to 100 kVoltspotential over a period of 40 to 100 nanoseconds. In still anotheralternative, a 1, 3, 9, or a 12 Volt battery, for example, can be usedto charge a capacitor to build voltage potentials of 100 volts to 1kilovolt which can be discharged across the primary coil of anon-contact electrode 12 to the tissue surface 15 or alternatively via aspark-gap regulator (see, FIGS. 4A and 5) to a single polarity tissuecontacting electrode 12. Finally, an alternating current source can beused in connection with a transformer to build the static voltagepotential and operate as disclosed herein in singular polarity form. Oneof ordinary skill in the electric arts will understand details of howappropriate circuitry can be arranged to use these and other electricenergy generators to build an appropriate voltage potential and to sendthe potential to the non-contact electrode 12 directly or first througha spark-gap regulator for added electrical discharge control.

In some embodiments where the electrodes are minimally invasive orcontact the surface of the tissue, the energy source can be one that iscapable of generating a sufficient electric voltage potential over aperiod of time less than 1 millisecond, and preferably less than 100microseconds. In preferred embodiments, the energy source is apiezoelectric crystal.

Using any of the different electric energy sources noted herein, thedischarge of the electric energy will occur naturally across thespark-gap regulator, or otherwise across the gap 17 from the non-contactelectrode 12 to the tissue surface 15, in an oscillatory fashion suchthat the polarity of the pulse actually reverses in nanosecond timeframes. Such discharge likely moderates the power intensity of the pulsethereby keeping the sparked voltage potential from burning, ablating orotherwise damaging the tissue surface 15.

FIGS. 4A and B are schematic drawings depicting examples of elements ofalternate designs for the device wherein the singular polarity electrodecomprises either a tissue contacting single polarity electrode array 19that is connected to a spark-gap regulator 18 as shown in FIG. 4A, oralternatively comprises a non-contact electrode 12 connected to aspark-gap regulator 18 as depicted in FIG. 4B.

With respect to the regulation of the total energies imparted to thetissue using the non-contact electrode, the energies sufficient forelectropermeabilization can be tailored by adjusting the measure of thegap between the tip of the non-contact electrode and the tissue surface,or alternatively the measure of the gap of a spark-gap regulator, oralternatively adjusting the combination of a gap in the spark-gapregulator within the circuitry and the gap between the tissue surfaceand non-contact electrode. As an example, the system electronics andelectrode can be arranged such that the gap between the non-contactelectrode and the tissue surface is not the only location for regulationof voltage discharge. Rather, the non-contact electrode can have aspark-gap regulator upstream from the non-contact electrode, as depictedin FIG. 4B, so as to set the discharge regulation away from thenon-contact electrode/treatment zone. This is similar to the alternatearrangement wherein the single polarity tissue contacting electrodearray is connected to a spark-gap regulator upstream as shown in FIG.4A.

Spark-gap regulation for electroporation allows for delivery ofextremely short and high intensity voltage pulses without danger to thetreated mammal and without macroscopically affecting the stratumcorneum. The minimal gap distance can be calculated for setting voltagesfor use in electroporation. For example, at 1 atmosphere (760 torr) androom temperature (20 degrees Celsius), based on Paschen's Law and theTownsend breakdown mechanism in gases, as understood by one of ordinaryskill in the art, where N is the density of air, “d” is the gapmeasurement, under the formula Voltage=K(Nd), the breakdown voltagenecessary to cross a gap of the measurements is disclosed in Table I.

FIG. 5 is a three dimensional view of a spark-gap regulator whereinelectric leads 22 and 23 are separated by a gap 24 of a predeterminedmeasurement inside housing 25. As shown in FIG. 5, the spark-gapregulator is a device comprising a housing 25, which is constructed ofan electrically inert material such as glass, Plexiglas or clearplastic, enclosing each of two wires, a lead wire 20 and a receivingwire 21 placed so that there is a gap 24 separating terminal ends 22 and23, respectively, thereof. The housing is constructed so that there canbe a vacuum space, if desired, comprising the gap 24. This vacuum aspectprovides for the allowance of any discharge of electric energy betweenthe terminal ends 22 and 23 of the wires across the gap to occur withoutatmospheric molecules influencing the resistance of electric chargetransfer across the gap. In this manner, the gap can be constructed tohave any length measurement practical for use in sparking charge fromthe lead to the receiving wire and thereby specifically controlling theamount of energy that can discharge across the gap. Typically, the gapcan measure between 0.01 and 4 cm.

FIGS. 6A, B, and C are graphs depicting the pulse characteristics ofelectrostatic discharges from various energy sources. FIG. 6A is a graphof the discharge from a Van de Graaff generator, FIG. 6B is a graph ofthe discharge from a piezoelectric crystal, and FIG. 6C is a graph ofthe discharge from a spark coil. Each of the Figures illustrates timevs. voltage.

As depicted in FIG. 6A, the discharge of a pulse from a Van de Graaffgenerator occurred across about 40 nanoseconds in a sinusoidal fashion,each polarity having a pulse of between about 2 and 5 nanoseconds. Thedischarge of a piezoelectric crystal provided a similar sinusoidaldischarge as shown in FIG. 6B. In this case, the bipolarity of thesinusoidal discharge occurred in a little over 10 nanoseconds. In stillanother example of electric pulse source discharge, a discharge acrossan air gap using a spark coil (step up transformer) is shown in FIG. 6C.In this instance, the discharge is even shorter on the order of 10nanoseconds but here, the discharge is in a single broad sinusoidalspike. Thus, the spark coil generated discharge has inherently longersingular pulses of either polarity. The longer the single pulse, themore association with damage to tissue is observed. In an embodiment,the Van de Graaff and piezoelectric generated potential and dischargeprovides superior results in causing no discernable effect on the tissuesurface while generation from a spark coil has the potential of beingassociated with effects on tissue if the total energy of the potentialis higher than a predeteminable level. Therefore, the sparks, howevergenerated, are those that can be generated and discharged into tissuesurface without either purposely or inadvertently causing damage to theintegrity of the stratum corneum.

FIGS. 7A to F are drawings of various electric circuits generally layingout the charge generation elements of alternate embodiments. In FIG. 7A,a Van de Graaff generator system is depicted wherein charge buildup isdelivered to the patient tissue directly with a non-contact singlepolarity electrode and a ground plate. This system can also include aspark-gap regulator if desired. In FIG. 7B, a battery power source canbe arranged to step up voltage and send charge through a spark-gapregulator to the patient tissue. In FIG. 7C, a simpler circuit isdisclosed wherein the charge generated is sent through a spark-gapregulator to the patient tissue. In FIG. 7D, a circuit wherein thecharge is generated using a piezoelectric crystal is disclosed. Here thecharge can be sent directly to the non-contact electrode or can be sentthrough a spark-gap regulator before being sent to the patient tissue.In FIG. 7E, a circuit is disclosed depicting a high voltage Field EffectTransistor (FET) switched coil. In FIG. 7F, a circuit is discloseddepicting a manually switched coil.

A Van de Graaff generator circuit can be relatively simple, using asbasic components a circuit comprising a Van de Graaff generator attachedto the non-contact electrode element such that the charge potentialgenerated is regulated in its delivery to the tissue by gapping acrossthe space between the non-contact electrode and the tissue surface.Alternatively, the charge potential can be sent to a spark-gap regulatorand then on to the tissue via either the non-contact electrode or thetissue contacting single polar array electrode. There need not be anycounter electrode. Rather, the system inherently provides for deliveryof energies that will discharge through the body tissue into theenvironment. An alternative to using no counter electrode can be the useof a ground plate or an electrically conductive foot pad to assistcomplete discharge of energy through the body. Further, given that thereis a lower net total charge imparted to the tissue via the Van de Graaffgenerated pulse (likely associated with the oscillatory nature of thestatic discharge, as shown in FIG. 6A), there is even less concern forneeding a counter electrode to dissipate the imparted electricpotential.

FIGS. 7B and C depict circuits that incorporate spark-gap regulators.These circuits are compatible with either the non-contact electrode orthe single polarity tissue contacting electrode. In FIG. 7B, batteries“Bat 1” and “Bat 2” are connected to the primary coils of “xfr 1” and“xfr 2” by switch SW1 wherein the secondary windings of xfr 1 and xfr 2are greater than their respective primary windings by the same ratiowith respect to the voltage in the secondary windings as compared to thevoltage of Bat 1 and Bat 2. These secondary windings are rectified bydiode D1, and connect to additional coils L1 and L2 which furtherincrease the voltage sufficient to jump the gap in the “spark-gap” unitwhich drives the primary coil of “xfr 3” to induce the final voltageseen by the spark electrode on the patient who is grounded to theenvironment. In this configuration, the spark-gap can be as little as0.01 cm. In FIG. 7C switch SW 1 transfers battery voltage to the primarycoil of xfrl and the secondary coil of xfr 1, which is larger than theprimary coil, and connects to a spark-gap regulator embedded within theprimary coil of the output transformer, thereafter leading to thepatient.

FIG. 7D is a drawing representing a circuit employing a piezoelectriccrystal. Like the Van de Graaff generator, the charge pulse can be sentdirectly to the non-contact electrode or can be sent through a spark-gapregulator to a single polarity tissue contacting electrode. Also, aswith the Van de Graaff generator, there need not be a return electrodebut alternatively a grounding means, such as a conductive pad forexample, on which the treated mammal stands can be used if desired.

FIGS. 7E and F depict coil voltage potential generator circuits. In FIG.7E a high voltage field effect transistor (FET) switched coil isdisclosed. Here, when a pulse is applied to the base of the FET 1 asdiagrammed by closing a switch, the coil L1 is switched to ground andcurrent begins to flow from the battery (Bat) through L1. When FET 1turns off, current tries to continue through the inductive coil of L1but cannot, and will trigger a spark through the patient until theenergy stored up in L1 dissipates. Similarly, FIG. 7F shows a manuallyswitched coil wherein the same mechanism occurs when the switch isopened after the coil has built up charge.

FIGS. 8A and B are graphs showing pulse discharges using a Van de Graaffgenerator. In FIG. 8A, the pulse is depicted wherein there are 100nanoseconds per division in the graph. There are about 500 nanosecondsof a diminishing pulse at about 1 Volt per division. This translates to10 Amps per division or a 25 Amp maximum current. This example has aninitial pulse spike lasting about 30 nanoseconds. In FIG. 8B, the samesection of the pulse is shown in 10 nanosecond increments showing theabout 30 nanosecond nature of the initial pulse spike.

In further related embodiments, as shown in the example of FIGS. 8A andB, the discharge from a Van de Graaff generator is actually anoscillating discharge wherein there is a reversal of the spark polaritywith reversals of charge flow in diminishing switchbacks until theenergy, as measured in volts, falls below that value allowing jumping ofthe charge across the gap. The oscillating charge flow across the gapcan occur lower than the initial gap cutoff value due to the charge flowacross the air gap becoming a plasma. The observed discharge thencontinues in oscillations running out to about 1 millisecond even thoughthe initial first pulse occurred in less than 40 nanoseconds.

In still other embodiments, the discharge of the electric potential canbe sent into the tissue using an alternate to the non-contactingelectrode, namely through a singular polarity tissue contactingelectrode. In this alternate embodiment, rather than a spark directly tothe tissue surface, the energy is directed to a single polarity array ofneedle-like projections after passing across an upstream spark-gapregulator. In an embodiment, the needle-like projections are either of anon-invasive type or alternately, of a minimally invasive type. Further,the singular polarity electrode can be fashioned from a simple singleblock of electrically conductive material. In a related embodiment, thearray of “pins” or needle-like projections can comprise an array ofvarious dimensions such as, for example, 1 to 100 pins arranged in agrid such as a 4×4 array or alternatively a 10×10, or any otherconfiguration or shape, such as a square grid or a circular grid.

In either alternate embodiment of the electrode, i.e., either thesingular polarity non-contact spark electrode, or the tissue contactingsingular polarity array, as one of skill in the electric arts willrecognize, there is a need for the voltage potential to dischargethrough the tissue and, ostensibly, find its way to a zero potential.This can be accomplished by either placing a discharge plate,essentially an opposite polarity electrode, in contact with the treatedmammal, or preferably, providing for the imparted charge to becomegrounded in the tissue of the treated animal itself. Not in contrastwith this physical need for the voltage potential to fully dischargefrom the site of entering the tissue, there is not a requirement forthere to be located an electrode of opposite potential near the site ofdelivery of the singular polarity discharge that is delivered from theelectrodes. The treated mammal should be grounded sufficiently to allowthe static charge imparted to the mammal to reach ground potential. Thiscan be accomplished by the imparted potential dissipating throughout themammal's body tissues, or alternatively, a remote electricallyconductive material can be placed in contact with the mammal's body,such as an electrically conductive foot pad.

For calculating the values in Table I, the majority of the discharge,whether using a Van de Graaff generator, a piezoelectric crystal, or aTesla coil, occurs at the front end of the pulse (as shown in FIGS. 8Aand B). Thus, taking the values obtained from the front end pulse spike,Amps, Coulombs, and total energy may be determined that is imparted intothe tissue occurring from a variety of starting voltage potentials. Forexample, a positive charge having a 20 Amp peak current and lastingapproximately 40 nanoseconds (Amps×pulse length=Coulombs) equalsapproximately 8.0×10E⁻⁷ Coulombs for a 10 mm gap. Each of the values inthe Table I were calculated similarly as one of ordinary skill in theart will understand.

TABLE I Total Net Minimum Representative Charge Energy KVolts to Currentfrom to Gap in breach gap Pulse Width Across gap electrode tissuemillimeters (breakdown) (nanoseconds) (Amps) (Coulombs) (Joules)  0.10.9 7 4 2.8 × 10E−8 0.000025  1 4.3 10 11 1.1 × 10E−7 0.00047  2 7.6 1215 1.8 × 10E−7 0.0014  5 16.4 29 16 4.6 × 10E−7 0.0076 10 (1 cm) 30.3 4020 8.0 × 10E−7 0.024 15 43.8 40 22 8.8 × 10E−7 0.039 20 (2 cm) 57.0 4029 1.2 × 10E−6 0.066 25 70.2 40 35 1.4 × 10E−6 0.098 30 (3 cm) 83.2 4538 1.7 × 10E−6 0.14 35 96.1 40 60 2.4 × 10E−6 0.23 40 (4 cm) 109.0 35 702.5 × 10E−6 0.27 Values based on 1 Atm (760 torr) at 20 degrees C, basedon Paschen's Law, Townsend breakdown mechanism in gases, V = K (Nd), N =air density, d = gap; Current measured with Pearson Model 411 CurrentMeter, calibrated at 0.1 V/Amp. Spark generated via Van de Graaffgenerator. Formula Q (Coulombs) = amps × t (seconds); E (Joules) = Q ×V.

Specifically, the minimum electric energy necessary to cross a gap of agiven distance using a Van de Graaff generator is disclosed in Table I.With that minimum voltage potential, the minimum pulse length and theminimum current can be calculated as disclosed. Further, the totalcharge, in Coulombs, discharged across the gap can be calculated. Thus,there is an ability to graph the actual discharge, and calculate the netenergy, in Joules, imparted into the tissue. From this Table I, theminimal energies capable of being delivered to the tissue surface forany given gap employed in the device can be determined whether theelectrode to tissue surface gap and/or the upstream spark-gap regulatoris used. This spark-gap regulation is useful for the alternateembodiments, e.g., the non-contact electrode and the tissue contactingelectrode array. In both instances, singular polarity voltage potentialdischarge can be administered with a known total energy delivery ondemand. Since that discharge will take place over a pulse period ofapproximately between 5 nanoseconds and 5 microseconds, there is nopotential harm to the treated mammal. This short pulse time periodallows for current of 4 to 70 Amps at very low total energies betweenabout 0.00001 and 0.5 Joules without any significant danger to thetreated mammal despite the high voltage levels associated with dischargeacross the gap.

For example, a Van de Graaff generator, such as for example, onechargeable to between 75 kVolts and 100 kVolts, was charged to 100kVolts and the charge discharged through the spark-gap was calculated tobe about 10⁻⁵ Coulombs. Specifically, as charge builds up on the largemetal head of the Van de Graaff generator, which acts as a capacitoracross the dielectric of air over a spark-gap of approximately 4 cm, abreakdown will occur over this gap once the voltage becomes over about100 kVolts. Upon the breakdown and charge beings delivery across thegap, air ionizes between the electrode head and the skin/tissue of themammal subject, and current begins to flow until the charge from the Vande Graaff equalizes with the static potential of the tissue. However,since the flowing current has inertia, the equal potential point betweenthe Van de Graaff head and tissue will reverse, and build up an oppositerelative charge as compared to the time when the breakdown initiated.There will be several oscillations of potential reversal as currentflows back and forth, until the potential difference drops below aminimum to keep the spark-gap air ionized, at which time the spark eventterminates.

In a similar fashion, calculations of energies imparted into the tissuecan be made for voltage potentials generated from both piezoelectriccrystals and Tesla coils as shown in Tables II and III, respectively,below. In these tabular calculations conditions for calculating were 1atmosphere (760 torr), 20 degrees Celsius, based on Paschen's Law,Townsend breakdown mechanism in gases; V=K (Nd), N=air density, d=gap;current measured with Pearson Model 411 Current Meter, calibrated at 0.1V/Amp. Q (Coulombs)=I (amps)×t (seconds), E (Joules)=Q×Volts.

TABLE II* Initial pulse Kvolts width Current Charge Energy Gap (mm)(breakdown) (nanosec) (Amps) (Coulombs) (Joules) 1 4.3 10 6 6.0 × 10E−80.00026 2 7.6 14 10 1.4 × 10E−7 0.0011 5 16.4 17 15 2.5 × 10E−7 0.004110 (1 cm) 30.3 30 18 5.4 × 10E−7 0.016 *Piezoelectric crystal generator

TABLE III** Initial pulse Kvolts width Current Charge Energy Gap (mm)(breakdown) (nanosec) (Amps) (Coulombs) (Joules) 1 4.3 13 2 2.8 × 10E−80.00012 2 7.6 14 2.5 3.5 × 10E−8 0.0026 5 16.4 15 6 9.0 × 10E−8 0.001510 (1 cm) 30.3 20 14 2.8 × 10E−7 0.0085 **Tesla coil generator

For example, calculations on total energy imparted were carried outusing a Pearson inductive current monitor (Model 411, PearsonElectronics, Inc., Palo Alto, Calif. USA) that develops a 0.1 Volt/Ampvoltage current ratio, and a Tektronix TDS210 oscilloscope (Tektronix,Inc., Beaverton, Oreg., USA) with a piezoelectric crystal that wascapable of generating a 1 cm spark length, resulted in the generation of25 Amps over a 20 nanosecond pulse to deliver about 10⁻⁶ Coulombs ofelectric charge. Specifically, I=dQ/dt with 1 Coulomb=1 Amp×1 Secresults in 25 Amps×20×10⁻⁹ sec=0.5×10⁻⁶ Coulombs.

In another example, using the formula I=delta Q/delta t, which can alsobe written as dQ=I×dt=V/R×dt, where I=current, Q=energy in Coulombs,V=volts, R=resistance in ohms, and t=time, using a piezoelectriccrystal, a current of 10 Amps is generated over a 100 nanosecond pulseand a total charge transfer of 10⁻⁶ Coulombs. Specifically, the tip ofthe piezoelectric generator, which is conductive, such as anelectrically conductive metal, and in contact with one side of thepiezoelectric crystal, is brought within about 1 cm of the skin/tissueof the mammal subject. The other side of the piezoelectric crystal isconnected to another electrical conduit that is subsequently grounded tothe environment of the subject mammal wherein the grounded subject is inelectrical communication with this second side of the crystal circuit.Upon mechanical impulse being applied to the crystal, a high voltagepulse between 15 and 35 kVolts is generated. This pulse, directed to theelectrode head, and positioned about 1 cm from the tissue will be at anelectrical potential with respect to the grounded subject that issufficiently high to ionize the air gap between the nearest point of thetest subject tissue surface to the electrode. Further, current will flowuntil the current built upon the crystal by the mechanical impulsedissipates.

As noted above, FIGS. 6A, B and C show the discharge of voltages fromthree different sources of power generation. In FIG. 6A, discharge froma Van de Graaff is shown wherein the discharge actually comprises anoscillating wave form. Thus, the total energy imparted to the tissue notonly occurs in less than about 40 nanoseconds, it is actually a net ofopposite polarities oscillating into the tissue. Further, the pulseperiod for each pulse polarity is extremely short, namely about 2 to 5nanoseconds. Similarly, as shown in FIG. 6B, discharge of apiezoelectric crystal naturally occurs in a somewhat oscillatoryfashion, again each polarity of the pulse being very short, about 2-5nanoseconds. Still further, as shown in FIG. 6C, the discharge from aspark coil also exhibits an oscillation in the actual discharge but inthis instance, the waveform comprises single long pulse periods ineither pole of the oscillation, a phenomenon different from thedischarge occurring from the Van de Graaff or piezoelectric crystalgenerators. The discharge from a spark coil generator, if too high avoltage potential, can cause tissue damage. Thus, when using a sparkcoil as a generation source, the system is tailored to discharge onlyenergies sufficient for electropermeabilization and not damage tissue.

Examples A. Non-Contact Electrode Experiment

A device comprising a Van de Graaff generator as a power source and anon-contact electrode was placed 1 cm above a mammalian tissue surface(guinea pig) was pulsed with either 4 or 8 spark discharges across the 1cm gap and above a 50 ul (microliter) bolus of previously deliveredintradermal injection of green florescent protein (2 mg/ml dna plasmidin PBS) expressing plasmid (plasmid pgw12-GFP from Aldevron, N. Dakota).The spark-gap used with the Van de Graaff generator is approximately 1cm, resulting in repeated sequence of 4 or 8 sparks of approximately 30kVolts, resulting in delivery of approximately 25 milliJoules of energyduring each spark event. If electropermeabilization has taken place andthe plasmid entered the cells in the epidermal tissue, the proteinencoded by the plasmid will be expressed and the green florescentprotein will fluoresce under UV light.

In this experiment, the non-contact electrode was placed directly abovethe site where a 50 ul bolus of GFP (2 mg/ml dna plasmid in PBS) wasinjected intradermally. The intensity of expression of the GFP issubstantial where only four spark discharges were administered. Similarresults were obtained where eight spark discharges were administered.

B. Single Polarity Non-Invasive Tissue Contacting Array

A device including a Van de Graaff generator and a non-invasive singlepolarity tissue contacting 4 by 4 array to deliver either 4 or 8 pulsesto tissue was obtained. Guinea pigs were obtained and treated in fourrepeat experiments by a 50 ul bolus intradermal injection of GFP (2mg/ml dna plasmid in PBS) followed by pulsing the tissue surface usingthe single polarity contact electrode (30 kVolts, gap of 1 cm, 4 to 8pulses of about 25 milliJoules energy). The pulses were effectivelypulsed to the array wherein each needle or pin received an equivalentcharge to dissipate into the tissue. The GFP was successfullyelectroporated into the epidermal tissues whether 4 pulses or 8 pulseswere administered.

C. Single Polarity Invasive Tissue Contacting Electrode

A device including a Van de Graaff generator and a tissue penetratingelectrode was tested in guinea pigs using 280 ul of 0.1 mg/ml GFPdelivered in a single bolus via delivery into the tissue from the tissuepenetrating electrode itself. The single spark onto an invasiveelectrode provided sites of electroporation to the tissue cells.

D. Electropermeabilization System Using Piezoelectric Crystal

A device comprising a piezoelectric crystal as a power source and acontact electrode array is placed 1 cm above a mammalian tissue surfaceand is pulsed with either 4 or 8 spark discharges across the 1 cm gapand above a 50 ul (microliter) bolus that is previously delivered byintradermal injection of green florescent protein (2 mg/ml dna plasmidin PBS) expressing plasmid (plasmid pgw12-GFP from Aldevron, N. Dakota).This gap should result in repeated sequence of 4 or 8 sparks, resultingin delivery of approximately 25 milliJoules of energy during each sparkevent. If electropermeabilization has taken place and the plasmidentered the cells in the epidermal tissue, the protein encoded by theplasmid will be expressed and the green florescent protein willfluoresce under UV light.

Application of the methods and devices described herein are well suitedfor cellular delivery of therapeutic molecules to cells for elicitingimmune responses or for other treatments. This technology is well suitedfor DNA based vaccine delivery and for gene-based therapies. Forexample, a therapeutic amount of substance comprising a polynucleotideencoding an antigen polypeptide or a formulation of the polynucleotideand biologic salts as one of skill in the pharmaceutical arts is wellversed, can be injected into epidermal, dermal, or subdermal tissuesfollowed by delivery to the tissue of a discharge of electric energyfrom the device via either the non-contact electrode or alternativelythe singular polarity tissue contacting array, and the injectedpolynucleotide will be electroporated into the cells of that tissue.Moreover, the use of the spark-gap method allows forelectropermeabilization of targeted tissue, specifically the upper mostlayers of the skin tissues. GFP expression only occurs in the top mostlayers of the skin.

Immune Experiment

In further experiments, animals injected with influenza antigen (NP) andpulsed using the apparatus, were studied for the expression ofantibodies to the antigen. Specifically, the test animals (guinea pigs)were given intradermal injections of the antigen (50 ul bolus comprising1 mg/ml NP plasmid) followed by electropermeabilization using thespark-gap apparatus (each animal receiving 10 sparks per injectionsite). Titers were followed out to ten weeks as shown in FIG. 9, whichis a graph showing titers of antibodies to influenza protein (NP)following dermal injection and pulsed using the spark-gap method. Thedata of the spark-gap pulsed animals is compared to immune responseelicited from delivery of antigen to muscle pulsed using a non spark-gapsystem, and to dermal injection of antigen without pulsing as control.The animals were boosted with an injection of antigen at week 4. Titersreached significant levels by week 5. These titers were superior tothose logged for muscle delivered and electroporated antigen.

While embodiments may have many different forms, there is shown in thedrawings and as herein described in detail various implementations withthe understanding that the present disclosure is to be consideredexemplary and is not intended to limit the invention to the embodimentsillustrated. The scope of the invention will be measured by the appendedclaims and their equivalents.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of various implementations, it will be apparent to those of skillin the art that variations may be applied to the compositions andmethods and in the steps or in the sequence of steps of the methoddescribed herein without departing from the spirit and scope of theinvention. More specifically, the described embodiments are to beconsidered in all respects only as illustrative and not restrictive. Allsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit and scope of the invention asdefined by the appended claims.

All patents, patent applications, and publications mentioned in thespecification are indicative of the levels of those of ordinary skill inthe art to which the invention pertains. All patents, patentapplications, and publications, including those to which priority oranother benefit is claimed, are herein incorporated by reference to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

The implementations illustratively described herein suitably may bepracticed in the absence of any element(s) not specifically disclosedherein. Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that use of such terms andexpressions imply excluding any equivalents of the features shown anddescribed in whole or in part thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by various implementations and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

What is claimed is:
 1. A method of enabling the delivery of therapeuticsubstances into cells of tissue of a mammal by using a sparkelectropermeabilizing device to deliver an electropermeabilizingelectric voltage potential to the surface of a mammalian tissue,comprising: generating a voltage potential sufficient to jump a gap ofpredetermined distance; delivering said generated voltage potential inthe form of a spark that can jump said gap to the surface of themammalian tissue without causing macroscopic damage to an integrity ofthe surface of the mammalian tissue; and delivering the therapeuticsubstances to cells within the mammalian tissue.
 2. The method of claim1, wherein the delivering step comprises: delivering the voltagepotential across a spark-gap regulator having a space gap; wherein thespaced gap is the gap of predetermined distance.
 3. The method of claim1, wherein the generated voltage potential is from 0.9 kV to 109 kV. 4.The method of claim 1, wherein the delivered voltage potential furthercomprises a total electric charge of about between 2.8×10E-8 and2.5×10E-6 Coulombs.
 5. The method of claim 1, wherein the deliveredvoltage potential imparts a total energy to the surface of a mammaliantissue about between 0.000025 and 0.27 Joules.
 6. The method of claim 1,wherein the delivered voltage potential has a pulse length of aboutbetween 5 nanoseconds and 5 microseconds.
 7. The method of claim 1,wherein the delivering step is repeated a multiplicity of times between2 and 20 pulses.
 8. The method of claim 1, wherein the therapeuticsubstance comprises a polynucleotide encoding an expressiblepolypeptide.
 9. The method of claim 1, wherein the therapeutic substancecomprises a polypeptide.
 10. The method of claim 1, wherein the energysource of the electric voltage potential is selected from one of a 1, 3,9 or 12V battery, a piezoelectric crystal, a charged induction coil, avan de Graaff generator, and a charged capacitor.
 11. The method ofclaim 2, further comprising: discharging from the electrode directly tothe surface of the mammalian tissue.
 12. The method of claim 2, furthercomprising: providing the spark-gap regulator having a housingcomprising an electrically inert material; providing a supply electriclead and a receiving electric lead that are each placed within thehousing and that are electrically isolated from one another; providing aspace gap between the supply electric lead and the receiving electriclead inside the housing; and adjusting an atmospheric pressure in thehousing to a predetermined value.
 13. The method of claim 12, whereinthe space gap has a measurement of between 0.01 cm and 4 cm.